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IntroductionNeurotrophins are soluble growth factors known mainly fortheir roles in regulating the development of the mammaliannervous system. Two types of receptors mediate the actionsof these polypeptides: the Trk family of tyrosine kinasereceptors and the p75 nerve growth factor (NGF) receptor(Bibel and Barde, 2000; Hempstead, 2002; Huang andReichardt, 2001; Kaplan and Miller, 2000). Gene targetingapproaches in mice have uncovered some functions of thesegenes in promoting survival and developmental maturation ofcertain types of neurons of the peripheral and central nervoussystem, thus confirming their critical role in neuraldevelopment (Huang and Reichardt, 2001). Furthermore, thephenotypes observed in these mutants have demonstrated thespecificity of the interactions between neurotrophins and theirTrk receptors. For example, both NGF- and TrkA-deficientmice have similar phenotypes. Ablation of either gene resultsin the virtual absence of the superior cervical ganglia neuronsby the first postnatal week, confirming the essential role ofNGF/TrkA signaling in the development of sympatheticneurons. Furthermore, both mutant mice are insensitive topain, because of the loss of small-diameter nociceptivesensory neurons (Huang and Reichardt, 2001; Tessarollo,1998).

It has long been hypothesized that neurotrophic factors,

particularly NGF, play a role in the development and functionof structures outside the nervous system (Levi-Montalcini,1987). Indeed, neurotrophins and their receptors are widelyexpressed in a variety of non-neuronal tissues throughoutdevelopment, including the cardiovascular, endocrine,reproductive, and immune systems (reviewed by Aloe et al.,1999; Tessarollo, 1998; Vega et al., 2003). However, ourknowledge of the function of neurotrophins in non-neuronaltissues is still fragmented. Evidence for such role was providedby mice lacking NT3 (Ntf3 – Mouse Genome Informatics)and BDNF, which revealed the requirement of these ligandsand their receptors for the normal development of thecardiovascular system (Donovan et al., 1996; Donovan et al.,2000; Tessarollo et al., 1997). In addition, neurotrophinfunctions outside the nervous system have been suggested fromphenotypes identified in hair follicles and thymus ofneurotrophin and Trk receptor mutant mice (Botchkarev et al.,2004; Garcia-Suarez et al., 2002; Garcia-Suarez et al., 2000).However, one can not exclude that some of these deficits wereindirect effects of the severe nervous system defects. Moreover,mice lacking neurotrophin functions, with the exception of p75and NT5 null mice, die soon after birth thus precluding analysisof phenotypes in the adult animal.

Within the immune system, p75 and TrkA receptors arewidely distributed and there is an increasing body of evidence

The nerve growth factor (NGF) receptor TrkA is widelyexpressed in non-neural tissues suggesting pleiotropicfunctions outside the nervous system. Based onpharmacological and immuno-depletion experiments, ithas been hypothesized that NGF plays an important role inthe normal development and function of the immunesystem. However, attempts to unravel these functions byconventional gene targeting in mice have been hamperedby the early postnatal lethality caused by null mutations.We have developed a novel ‘reverse conditional’ genetargeting strategy by which TrkA function is restoredspecifically in the nervous system. Mice lacking TrkA innon-neuronal tissues are viable and appear grossly normal.All major immune system cell populations are present in

normal numbers and distributions. However, mutant micehave elevated serum levels of certain immunoglobulinclasses and accumulate B1 cells with aging. These data,confirmed in a classical reconstitution model usingembryonic fetal liver from TrkA-null mice, demonstratethat endogenous NGF modulates B cell developmentthrough TrkA in vivo. Furthermore, they demonstrate thatmany of the dramatic effects previously reported bypharmacological or immuno-depletion approaches do notreflect physiological developmental roles of TrkA in theimmune system.

Key words: TrkA, NGF, Immune system, Mouse

Summary

Ablation of TrkA function in the immune system causes B cellabnormalitiesVincenzo Coppola 1, Colleen A. Barrick 1, Eileen A. Southon 1, Arkady Celeste 2, Kathryn Wang 3, Bei Chen 3,El-Bdaoui Haddad 3, Jian Yin 3, Andre Nussenzweig 2, Arun Subramaniam 3 and Lino Tessarollo 1,*1Neural Development Group, Mouse Cancer Genetics Program, NCI, NIH, Frederick, MD 21702-1201, USA2Experimental Immunology Branch, NCI, NIH, Bethesda, MD 20892, USA3Aventis Pharmaceuticals, Bridgewater, NJ 08807-0800, USA*Author for correspondence (e-mail: tessarol@ncifcrf.gov)

Accepted 2 August 2004

Development 131, 5185-5195Published by The Company of Biologists 2004doi:10.1242/dev.01383

Research article

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suggesting that NGF can regulate immune cell functionsincluding inflammatory responses (Aloe et al., 1999; Miceraet al., 2003; Tessarollo, 1998; Vega et al., 2003). For example,NGF blood levels are increased in several autoimmune andallergic human diseases (Aloe et al., 1997; Aloe et al., 1994).Furthermore, NGF can accelerate wound healing bymodulating the inflammatory response in mice (Matsuda etal., 1998; Micera et al., 2001). However, the most intriguingdata concern the effects of NGF on B-cell (Brodie andGelfand, 1994; Brodie et al., 1996; Otten et al., 1989) andmast cell function (Leon et al., 1994). NGF can regulateimmunoglobulin (Ig) production in vitro and serves as asurvival factor for memory B cells (Brodie and Gelfand,1994; Brodie et al., 1996; Torcia et al., 1996). Furthermore,NGF is synthesized, stored and released by mast cells(Leon et al., 1994). Yet, the physiological rather thanpharmacological relevance of these effects is not clear(Tessarollo, 1998).

P75 mutant mice are viable and therefore amenable toinvestigation of non-neuronal structures including the immunesystem in the adult (Lee et al., 1992). However, a recentinvestigation has not found major deficits in the spleen andthymus of p75 mutant mice (Garcia-Suarez et al., 2001)and our own preliminary analysis has failed to unveil anyovert immunological deficits (V.C. and L.T., unpublished).Therefore, we hypothesized that TrkA mediates most of theNGF functions described in the immune system likewise in thenervous system. To test this hypothesis in vivo, we decided togenerate and characterize mice with a conditional null allele ofthe TrkA gene.

The classic approach to study the function of a gene in aspecific organ includes the generation of a mouse strain withloxP sites flanking the gene of interest (floxed) and anothermouse strain with the Cre recombinase under thetranscriptional control of a promoter expressed in the organ ofinterest. By crossing these two strains, deletion of the geneflanked by loxP sequences should be achieved in an organsite-specific manner. However, one major limitation of thisapproach is the fact that deletion of the floxed gene is notalways complete in all cells where Cre should be active(Rickert et al., 1997). Thus, the residual activity of the genecan potentially confound the analysis. This is particularlycritical for the immune system in which clonal expansion ofcells with a functional floxed allele may compensate forcertain phenotypes. To obviate this problem we have reversedthe strategy and activated TrkA specifically in neuronalstructures within a null mutation background. This approachprovides a means to distinguish between an intrinsic role ofTrkA in the immune system versus defects that are caused bynervous system deficiencies in the complete knockout mousemodel.

Using this strategy, we found that on the whole, the TrkA-deficient immune system develops normally. This resultdemonstrates that many of the dramatic effects previouslyreported by pharmacological or immuno-depletion approachesdo not reflect physiological roles of TrkA in the immunesystem during homeostasis. However, we also identifieddisregulation of Ig production and accumulation of a B-cellsubset. These data are the first demonstration that NGF/TrkAindeed modulates immunological functions not mediated bythe nervous system in vivo.

Materials and methodsMiceTrkAneo/neomice were generated by a gene targeting approach usingthe CJ7 embryonic stem cell line as previously described (Bonin etal., 2001; Tessarollo, 2001). The nervous system-specific Tα1-Cretransgenic mouse line was generated by microinjection of fertilizedeggs (Osborn et al., 1987) of a vector generated by fusion of the Tα-1 promoter region (a gift from Freda Miller) with the coding sequenceof the Cre recombinase and the SV40 polyadenylation signal (Glosteret al., 1994). TrkA-deficient mice used for the reconstitutionexperiment were previously described (Liebl et al., 2000).

The Rosa-26 reporter mouse strain was kindly provided by PhilSoriano (Soriano, 1999). Adult C57Bl/6-SCID mice (Bosma et al.,1983) used in reconstitution experiments were purchased fromCharles River (Frederick, MD, USA) and reconstituted by tail-veininjection with 5×106 E14.5 fetal liver cells.

All animals were treated in accordance with the guidelines providedby the Animal Care and Use Committee of the National CancerInstitute at Frederick (MD, USA).

Immunization protocolsTo test T-cell-dependent (TD) humoral responses, 3-6-month-old micewere subjected to retro-ocular bleeding to determine basal Ig levelsand then were injected intraperitoneally with 100 µg KLH-TNP(keyhole limpet hemocyanin-2,4,6,-trinitrophenyl; BiosearchTechnologies, Novato, CA, USA) in PBS (day 0). After 40 days, micewere given a boosting dose of the same Ag and blood was drawn againafter 1 week (day 47) and 2 weeks (day 54) from the boost. To testT-cell-independent (TI) type I and type II responses, mice were pre-bled and immunized with 20 µg of lipolysaccaride (LPS)-TNP or 25µg of Ficoll-TNP (Biosearch Technologies), respectively. Blood wascollected one and two weeks after challenge.

Northern blot and RT-PCRTotal RNA was isolated from different tissues by a single-step method(RNA STAT-60, Tel-Test ‘B’, Friendswood, TX, USA) and analyzedby classical northern blot analysis. TrkA transcripts were detected byusing a rat kinase-specific TrkA probe (spanning exon 12 to exon 17).RT-PCR was performed using SuperScript™ RT-PCR System(GIBCO-Invitrogen, Grand Island, NY, USA) according tomanufacturer instructions with the following primers: TrkA Exon10-Exon14 fragment: F primer: 5′-ACG GTA ACA GCA CAT CAAGAG-3′; R primer: 5′-CGG AGG AAA CGG TTG AGG TC-3′. β-actin (F primer: 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′; R primer: 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′).

Histological proceduresSections (5 µm) from paraffin-embedded tissues were stained withanti-CD45R, 1:200 (B220, RA3-6B2 BD-Pharmingen, San Jose, CA,USA), anti-CD3, 1:600 (Dako, Carpinteria, CA, USA), and anti-mouse F4/80, 1:25 (Caltag, Burlingame, CA, USA) for B-cell, T-celland macrophage-specific immunostaining, respectively. Apoptoticcells were detected with the Apoptag kit (Intergen, Purchase, NY,USA) following the manufacturer’s recommendations. For β-galactosidase staining, E11.5 embryos were fixed in PBS withoutMg2+/Ca2+, 1% formaldehyde, 0.2% glutaraldehyde, 0.02% NP-40 for30 minutes at 4°C, washed twice in PBS without Mg2+/Ca2+ at RT for20 minutes, and stained overnight at 30°C with PBS withoutMg2+/Ca2+, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, and1 mg/ml X-gal. For adult tissue staining, 20 µm frozen sections werepost-fixed with 4% phosphate-buffered paraformaldehyde for 5minutes, washed three times for 5 minutes with 100 mM sodiumphosphate buffer, 20 mM MgCl2, 0.01% sodium deoxycholate, 0.02%NP-40, then stained overnight as above, rinsed in dH2O andcounterstained with neutral red for 40 seconds. After three rinses in100% ethanol, cover slips were mounted in xylene.

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5187TrkA function in the immune system

Automated blood cell counts were performed using theHEMAVET 850 apparatus (CDC Technologies, Oxford, CT, USA).

FACS and ELISA Ig analysisFor FACS analysis, samples were prepared as previously described(Coppola et al., 1998). All the unlabelled antibodies, phycoerythrin(PE), fluorescein isothiocyanate (FITC) or PerCP-conjugated werepurchased from BD Pharmingen (San Jose, CA, USA). Elisa Iganalysis was performed using a modified protocol from BDPharmingen. Briefly, plates (Dynex-Immulon®) were coated overnightwith capture anti-Ig-specific Ab in PBS, washed once with PBS-0.05% Tween® 20, blocked for 1 hour at RT with PBS-3% BSA andincubated with serial dilutions of sera overnight at 4°C. The followingday, the plates were washed five times and incubated with a secondaryAb Ig conjugated to HRP diluted 1:1000 for 1 hour at RT. After sixfurther washes, substrate (ABTS Microwell Peroxidase SubstrateSystem, Gaithersburg, MD, USA) was added. Capture mouse anti-IgM, anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG3, anti-IgA, and theHRP-conjugated Abs were purchased from Southern Biotech(Birmingham, AL, USA). All standards for the different Ig classes,capture anti-IgE, and biotin-conjugated anti-IgE were purchased fromBD Pharmingen. For the IgE Elisa, HRP-streptavidin was purchasedfrom Jackson ImmunoResearch (West Grove, PA, USA). For anti-TNP-specific Ig dosage, the plates were coated overnight at 4°C withOVA-TNP (Biosearch Technologies) at 10 µg/ml in carbonate/bicarbonate buffer pH 9.6. The following steps were similar to thedosage of total sera Ig. Plates were read using a Benchmark Bio-RadElisa reader at 405 nm wavelength (Software: Microplate ManagerIII , Bio-Rad, Hercules, CA, USA). Data were analyzed and plottedusing GraphPad PRISM 3.0 (GraphPad Software, San Diego, CA,USA).

Passive cutaneous anaphylaxisIgE-dependent passive cutaneous reaction was generated bysensitizing the skin with an intradermal injection of anti-dinitrophenyl(DNP) IgE, followed 18-20 hours later by an injection of DNP-albumin (100 µl of a 1 µg/µl solution in saline) into the mouse tailvein. The right ear was injected intradermally with 25 ng of anti-DNPIgE and the left ear received 25 µl of saline to serve as control. Earthickness was measured with a caliper (Dyer Company, Lancaster, PA,USA) 15 minutes before and 15 minutes after the antigen challenge.

Generation of bone marrow derived mast cells anddegranulation assayBone marrow derived mast cells (BMMC) were generated byculturing femoral bone marrow cells from 6- to 8-week-old mice.Mice were sacrificed by CO2 asphyxiation and intact femurs wereremoved. Bone marrow cells were harvested by flushing the boneshaft with DMEM (GIBCO-Invitrogen) medium with 2% FBS. Redblood cells were lysed by incubation with 0.8% ammonium chloridewith 0.1 mM EDTA. Bone marrow cell cultures were established at adensity of 1×106 cells/ml in DMEM containing 20% FBS, 100 U/mlpenicillin, 100 µg/ml streptomycin, 50 ng/ml recombinant mousestem cell factor (SCF), 10 ng/ml recombinant mouse IL-3. Non-adherent cells were transferred to fresh culture plates once a week andfed by replacing 50% of the medium twice a week. After 4 to 6 weeksof culture, the purity of BMMC culture was greater that 95% asconfirmed by FACS analysis for FcεRI and c-kitexpression. BMMCwere activated via FcεRI stimulation by first sensitizing overnightwith anti-DNP IgE antibody (Sigma Aldrich, St Louis, MO, USA) at1 µg/ml in culture medium. Sensitized cells were washed andstimulated for 30 minutes at 37°C with 100 ng/ml DNP conjugate ofhuman serum albumin (DNP-HSA) (Sigma) in Hanks’ Balance SaltSolution supplemented with 2 mg/ml glucose and 0.03% BSA. Cellsupernatants were harvested and the cell pellets were lysed by freeze-and-thaw cycle. β-hexosaminidase was quantified in the supernatantsand cell lysates by spectrophotometric analysis of hydrolysis of

p-NAG (p-hitrophenyl-N-acetyl-D-glucosaminidine) (Sigma). β-hexosaminidase release was calculated as the percentage of β-hexosaminidase presented in the supernatants relative to the totalamount of β-hexosaminidase present in the supernatant and cell pellet.

ResultsMice lacking the TrkA receptor in non-neuronaltissues are viable and display no overt phenotypeNGF and TrkA knockout mice have similar phenotypes.Ablation of either gene results in severe losses of peripheralsensory and sympathetic neurons and early post-natal lethality(Crowley et al., 1994; Smeyne et al., 1994). Even though TrkAis widely expressed outside the nervous system, the earlydemise of these mutant mice has been attributed to the severeneuronal deficits. We hypothesized that restoration of TrkAfunction in the nervous system would rescue the lethalityassociated with the general deletion of TrkA and would allowus to investigate TrkA-mediated NGF functions in the manynon-neuronal organs in which its expression has been reported(Aloe et al., 1999; Tessarollo, 1998; Vega et al., 2003). To thisend, we devised the following ‘reverse conditional knockout’strategy. By gene targeting, we first generated an allele thatharbors a loxP-flanked neomycin resistance gene in intron 11,upstream of the tyrosine kinase encoding exons (TrkAneo/neo).Mice with the neo gene intact within the TrkAlocus develop aphenotype identical to that of TrkA null mice (data not shown)(Smeyne et al., 1994). Northern blot and RT-PCR analysisverified that no RNA capable of producing a functional TrkAprotein is generated by the mutant locus (Fig. 1C,D and datanot shown). Thus, the insertion of the loxP-flanked neo-containing cassette into an intron of the TrkA locus disruptsexpression of the gene creating a true null mutation. However,removal of the neo cassette by ubiquitous Cre-mediatedrecombination can restore TrkA function and rescues thelethality demonstrating that the residual loxP site does notinterfere with TrkAexpression (TrkAloxP/loxP) (Fig. 1A,C). Wethen developed a transgenic mouse line using the Tα-1 tubulinpromoter (Gloster et al., 1994) to express the Cre recombinaseexclusively in the nervous system (Tα-1-Cre) (Fig. 1E,F). Tα-1-Cre mice do not show any obvious phenotype and they areindistinguishable from non-transgenic littermate controls (notshown). To confirm that Tα-1-Creexpression is specific to thenervous system and no ectopic cre expression was present inorgans of the immune system, we crossed the Tα-1-Cretransgenic mouse to the Rosa-26 reporter strain (Soriano,1999) (Fig. 1E). β-galactosidase staining of several lymphoidorgans failed to detect any Cre recombinase activity (Fig. 1F),confirming the specificity of the Tα-1-Cre transgenic mouseline.

Crossing of the Tα-1-Creallele into the TrkAneo/neomutationgenerated mice that were viable and showed no overtphenotype. However, the presence of the neo cassette outsidethe nervous system completely inactivates TrkA (Fig. 1C,D)and thus allows the investigation of non-neuronal tissueslacking this gene function. Lack of neo-excision in several non-neuronal organs including spleen, thymus, bone marrow andlymph nodes of mice was further confirmed by PCR analysis(not shown). These data provide definitive evidence that TrkAfunction in the nervous system is the cause of the lethalityobserved in the TrkA knockout mice.

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TrkA is dispensable for the development of immunesystem organsOver the years, the NGF/TrkA ligand/receptor system has beenextensively implicated in regulating the development andfunction of the immune system. However, the lack of suitablein vivo models has hampered the investigation toward thedissection of a direct role of NGF/TrkA in this system.For example, systemic knockouts or models based onimmunodepletion of endogenous NGF affect all organsincluding the nervous system, which may be required fornormal development of the lymphoid organs. Our modelinactivates the NGF receptor TrkA only in non-neuronal tissuesand thus allows us to investigate the intrinsic function of this

receptor in structures including the organs of the immunesystem. Anatomical and histological analysis of thymus, spleenand lymph nodes did not reveal any significant differencebetween TrkA-deficient and control tissues. For example,cellularity and morphology of the spleen of mutant mice weresimilar to control littermates (not shown). In addition,lymphoid follicles (Fig. 2A,B) and distribution of apoptoticcells (Fig. 2C,D) were also present in similar numbers. Spleensections immunostained with the B-cell-specific B220 and T-cell-specific anti-CD3 antibodies did not detect any differencein number or distribution of B- and T-cells in the Tα-1-Cre;TrkAneo/neomutants compared with control littermates.(Fig. 2E-H). Similarly, lymph nodes, thymus and bone marrow

Development 131 (20) Research article

Fig. 1.Conditional inactivation of TrkA. (A) Diagram of the replacement vector strategy used to generate the TrkAneoallele and re-activation ofTrkA expression by Cre recombinase. The pGKneobpA cassette flanked by loxP sites was placed into intron 11. (B) Southern blot analysis oftail DNA from three-week-old mice with a 5′external probe shows the switch of the wild-type (WT) BamHI fragment from 12 kb (WT) to a 7kb restriction fragment (TrkAneo) after neo insertion. (C) Northern blot analysis of total RNA extracted from Dorsal Root Ganglia (DRG)neurons of E13.5 embryos either WT (+/+), heterozygous for the neo insertion (+/neo), homozygous for the neo insertion (neo/neo), orhomozygous for the allele after neo removal with β-actin cre (loxP/loxP), hybridized with a TrkA-kinase domain-specific probe (exon 14-17).Note that neo insertion completely eliminates TrkA-specific transcripts whereas TrkA-transcription is restored to WT levels after neo-excisionby Cre recombination. (D) RT-PCR analysis of the same samples analyzed by northern blot. Specific primers for actin, neo, and TrkA exon 10and 14 were used. Samples were treated with (+) and without (–) RT. Note the complete absence of the 609 bp TrkA-specific PCR product inTrkAneo/neomice. (E) Wholemount lacZstaining of a Rosa-26 E11.5 embryo with theTα1-cre transgene. Note the specific staining in bothcentral and peripheral nervous system. (F) lacZstaining of sections from spleen, thymus and bone marrow of adult Rosa-26 mice carrying theTα1-Cre(top panels) or a β-actin-Cre transgene with mosaic expression used as control (bottom panels) (Ma et al., 2003). Cre recombinaseunder the control of the Tα-1 promoter is only expressed in the nervous system (E) and not in organs of the immune system (F).

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seemed unaffected by TrkA loss at the anatomical andhistological level (data not shown). To assess the major cellpopulations of the immune system organs, we performedcytofluorimetric analysis on thymus, spleen and bone marrowcells with anti-B220, -IgM, -IgD, -CD19, -CD3, -CD4,-CD8, -Mac-1, -Gr1 and -CD5 antibodies. No quantitative orqualitative abnormalities in the B- and T-cell populations,macrophages and granulocytes (not shown) were detected inmutant mice. Furthermore, blood cell counts of mutant micewere also comparable to littermate controls (Table 1).

Taken together these data provide definitive evidence thatTrkA-mediated NGF function is dispensable for normaldevelopment of the immune system. Furthermore, thesefindings suggest that immune system deficiencies reported inother models are most likely because of indirect effects causedby NGF/TrkA loss in the nervous system.

Mast cells develop and function in TrkA mutant miceMast cells constitute another cell population for which thereis ample literature suggesting a direct role of NGF in theirdevelopment and function (Aloe et al., 1999; Tessarollo,1998; Vega et al., 2003). Toluidine blue and alcian blue/safranin staining of histological preparations from skin,spleen and lymph node of Tα1-Cre;TrkAneo/neomice showedthat mast cells were present in comparable numbers anddistribution to that of tissues from control littermates (Fig.2L,M and data not shown). Thus, TrkA signaling is notrequired for mast cell differentiation in vivo. To begin toaddress whether mast cells lacking TrkA displayed functionalabnormalities in vivo, we employed a wheal and flareparadigm. The skin of ears from mutant and control mice wassensitized with an intradermal injection of anti-DNP IgEfollowed by a systemic injection of DNP-albumin, asdescribed. Compared with saline challenged skin, IgE/DNPintradermal injections caused prominent mast cell

degranulation and mixed inflammatory cell infiltrates in bothmutant and wild-type mice (Fig. 3A-F). However, nosignificant differences were observed in mast celldegranulation activity in the two genotypes (Fig. 3 and datanot shown). It should be noted that this result does notpreclude the presence of subtle defects caused by TrkA lossbecause we have observed great variability in the responseamong individuals of both control and mutant group. Inaddition, on the current genetic background, the responseto DNP by mutant and control mice was approximately 20-fold lower than that of mice on a pure Balb/c background,making it difficult to assess the presence of subtle phenotypesin vivo (data not shown). Nevertheless, the in vivodegranulation response of mast cells lacking TrkA wouldsuggest that this gene is dispensable for mast cells’ activity.Because in vivo mast cells develop normally and can beactivated by passive cutaneous reaction, we then derived mast

Fig. 2.TrkA deficiency does not affect spleen development and skin mast cells. Spleens of wild-type (A,C,E,G) and mutant (B,D,F,H) micewere stained with hematoxylin/eosin (A,B), Apoptag (C,D), and B cell- (anti-B220; E,F) and T cell-specific (anti-CD3; G,H) antibodies. Notethe absence of any remarkable difference between mutant and control organs. (L,M) Toluidine-blue staining of mutant (M) skin samples showspresence of mast cells (arrows) in number and distribution similar to wild-type littermate controls (L). LF, lymphoid follicle.

Table 1. TrkAneo/neomice have a normal hematologicalprofile

WBC RBC PLT(103/µl) % Ly % Ne % Mo (106/µl) (103/µl)

MalesCTR 9.04±1.5 78.2±4.5 17.7±4.9 3.6±1.8 10.1±0.8 643±144MUT 9.31±1.4 75.5±9.9 20.2±10.0 3.7±1.2 9.9±0.6 592±72

FemalesCTR 9.14±1.4 84.6±1.7 10.2±2.6 4.9±1.9 9.3±0.7 558±128MUT 8.86±1.6 82.4±2.8 13.2±2.9 3.9±1.9 9.3±0.3 643±145

Absolute numbers of leukocytes (WBC), and erythrocytes (RBC) andplatelets (PLT) in TrkAneo/neo mice (MUT) do not show any significantdifference when compared with control littermates (CTR). In addition,percentages of blood lymphocytes (Ly), neutrophils (Ne) and monocytes (Mo)are not significantly different in either male or female TrkAneo/neoanimalscompared with controls.

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cells from bone marrow (BMMC) of TrkA mutant and controlmice to investigate whether loss of TrkA had any effect at allon the differentiation and functional potential of this cellpopulation. No apparent differences were observed in theircapacity to differentiate in vitro. However, when BMMCwere induced to degranulate by IgE cross-linking, the TrkA-deficient BMMC showed a modest but significant reductionin β-hexoseaminidase release in comparison with BMMCderived from control mice (P<0.01) (Fig. 3G). This resultsuggests that, although TrkA is not required for mast cell

development in vivo, its loss may nevertheless impact theirnormal activity.

TrkA deficiency causes abnormal Ig serum levelsWe found that lack of TrkA-mediated NGF signaling iscompatible with the development of lymphoid organs.However, it has long been suggested that NGF signaling mayalso modulate specific cell functions. For example, in vitroexperiments have shown that NGF can regulate Ig production(Brodie et al., 1996; Kimata et al., 1991a; Kimata et al., 1991b;

Development 131 (20) Research article

Fig. 3.Mast cells degranulatein TrkA-deficient mice.Histological assessment ofpassive cutaneous anaphylaxisin control (A,C,E) and TrkAmutant (B,D,F) mouse earsections stained for naphtholAS-D chloroacetate esterase.Compared with salinechallenged mice (A,B), theIgE/DNP-treated mice (C-F)exhibited prominent mast celldegranulation and mixedinflammatory cell infiltrates.Arrows are directed todegranulated mast cells. (G) β-hexoseaminidase release frombone marrow-derived mastcells obtained from TrkAmutant (TrkAneo/neo) andcontrol (TrkA+/+) littermatemice was measured asdescribed in Materials andmethods. Mast cells obtainedfrom the TrkA mutant miceshow reduced β-hexoseaminidase releaserelative to those from controls(*P<0.01).

5191TrkA function in the immune system

Kimata et al., 1991c). Indeed, serum analysis of Igs in Tα1-Cre;TrkAneo/neomice revealed a specific increase in the levelsof IgM, IgG2a and IgG1 (Fig. 4A). In contrast, IgG2b, IgG3,IgA and IgE (Fig. 4A and data not shown) were notsignificantly different from control littermates. Because thesedifferences, albeit significant, were small, we next comparedthis genetic model with the ‘classical reconstitution model’(Schlesinger et al., 1965). The immune system of adultC57Bl/6-SCID mice was reconstituted with fetal liver cellsfrom a previously generated TrkA null mutant mouse line(Liebl et al., 2000). Reconstitution efficiency was monitoredmonthly by flow cytometry analysis of peripheral blood B- andT-cells. Mice reconstituted with TrkA-deficient cells showedthe same B- and T-cell numbers compared with animalsreconstituted with littermate wild-type cells (not shown). Asobserved in the ‘reverse conditional TrkA mouse model’, wefound increased Ig levels in mice reconstituted with TrkA-deficient fetal cells (Fig. 4B). Importantly, these data validateour genetic model. Although the match is not perfect, perhapsbecause the stimulation condition is different in the two

models, it remains interesting that both systems show anincrease in Ig production, suggesting that overall, NGF exertsan inhibitory role on Ig production through TrkA in vivo.Therefore, detailed characterization of subtle phenotypes canbe more appropriately studied in a genetic system that is lesssusceptible to the variations associated with reconstitutionexperiments.

TrkA deficiency does not impair B-cell memory andT-cell independent responsesBecause it has been reported that NGF is a survival factor formemory B cells (Torcia et al., 1996), we next investigated Bcell memory response in the absence of TrkA. Mice wereinjected with KLH-TNP [T-cell-dependent (TD)-antigen] andmemory response was elicited after six weeks with a boostinjection of the same antigen. Specific anti-TNP Ig were dosedone and two weeks after the boost. Mice lacking TrkA in theimmune system produced Ig against the TNP antigen at evenhigher levels than controls, at least for the IgG2b class (Fig.

Fig. 4.TrkA deficiency in the immune system causes increased Igproduction. (A) Tα1-Cre;TrkAneo/neomice have increased levels ofspecific classes of Igs. IgM, IgG2a, IgG2b, IgG1 and IgG3 serumlevels were measured by ELISA in 12-16-week-old mutants (KO,n=18) and control littermates (CTR, n=14). Data are expressed inUnits/ml where 1 U=1 mg for IgM and 1 U=1 µg for IgG2a, IgG2b,IgG1 and IgG3. Bars: means±s.e.m. Data were evaluated using atwo-tailed Student’s t-test. *P<0.05. (B) C57Bl/6-SCID micereconstituted with TrkA-deficient fetal liver cells (KO, n=10) haveincreased levels of serum Igs compared with TrkA wild-type-reconstituted controls (WT, n=6). C57Bl/6-SCID mice werereconstituted and monitored for Ig serum levels from 1 to 5 monthsafter reconstitution. Data after two months from reconstitution areshown. All Ig classes are higher in TrkA-deficient animals, althoughonly IgM and IgG2b are statistically different from controls (two-tailed Student’s t-test. *P<0.05).

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Fig. 5.Absence of TrkA does not impair T-cell-dependent memorynor T-cell-independent type I and II B responses. (A) For the TDmemory response, Tα1-Cre;TrkAneo/neo(n=7) and control littermates(n=6) were immunized with KLH-TNP as described in Materials andmethods. (B,C) for the TI type I and II responses, mice wereimmunized respectively with LPS-TNP (B, six mutants and fivecontrols) or Ficoll-TNP (C, six mutants and seven controls). Serumlevels of specific anti-TNP Igs were measured by ELISA beforeimmunization (0), at one (1) and two (2) weeks after the challenge.Bars: means±s.e.m. Data were evaluated using a two-tailed Student’st-test. *P<0.05.

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5A). Therefore, TrkA-deficient B cells can mount a humoralmemory response. Next, we investigated whether the T-cell-independent (TI) type I and type II B-cell responses wereaffected by the TrkA deletion. Mice were immunized withLPS-TNP for the TI type I response or Ficoll-TNP for the TItype II response (Hasan et al., 2002). Again, anti-TNP-specificIg levels in mutant mice after one and two weeks from theboost injection were comparable or even higher than those ofcontrol littermates, indicating that TI responses are mountedefficiently in the absence of TrkA (Fig. 5B,C). Instead, thehigher levels of TNP-specific IgM observed in the absence ofTrkA following LPS immunization suggest that the abnormalIg production in the mutant mice is B-cell autonomous.

TrkA deficiency leads to accumulation of B1lymphocytesThe increased Ig levels observed in TrkA mutant mice suggestthe presence of B-cell abnormalities. Changes in Ig levels havealso been observed in mice with defects in B Cell Receptor(BCR) signaling (Hardy and Hayakawa, 2001). In vivoalterations in BCR signaling are often associated with changesin B1 lymphocyte numbers (Hardy and Hayakawa, 2001;Hayakawa and Hardy, 2000). Thus, we used flow cytometry toanalyze the peritoneal B1 cell population of four-, eight- andtwelve-month-old mice. The absolute number of cellsrecovered from the peritoneal cavity was similar in mutant andcontrol mice (data not shown). Four- and eight-month-oldmutant mice harbored normal B1 cell numbers (data notshown). However, 12-months or older mutant mice (n=7)showed a 30% (P<0.05) increase of B1 cells compared withcontrol littermates (n=6) (Fig. 6). This was demonstrated by anincreased population of lymphocytes expressing B220low,IgMhigh, Mac-1+, IgDlow and CD5+, which represents thehallmark of B1 lymphocytes (Fig. 6) (Hardy et al., 2000;Hayakawa and Hardy, 2000). These data suggest that TrkAmay control lymphoid function by modulating BCR signaling.

DiscussionA role for NGF in the normal development of the immunesystem has long been hypothesized and yet there is noconvincing data supporting this function in vivo. A majorlimitation in addressing this issue has been the lack of asuitable in vivo model to dissect direct versus indirect effectsof NGF on lymphoid organs. For example, systemic ablationof NGF or its TrkA receptor causes such severe sensory andsympathetic neuronal losses that phenotypes observed in non-neuronal organs could be caused by innervation deficits ratherthan an intrinsic loss of NGF/TrkA function. We havegenerated a model that provides a means to address thisquestion by rescuing TrkA function in the nervous system, butnot in non-neuronal structures. We have shown that NGF/TrkAis dispensable for the development of the lymphoid organs andyet causes defects including increased Ig production andaccumulation of peritoneal B1 cells. These data are consistentwith a modulatory role of this ligand/receptor system onimmunological functions.

The phenotype observed in the mutant mouse used in ourstudy is less severe than the one observed in the completeTrkA-deficient mice which have thymus abnormalitiesconsistent with organogenesis defects (Garcia-Suarez et al.,

2000). Because the difference between these two mutants ismainly the presence (this study) or the absence (Garcia-Suarezet al., 2000) of a fully developed nervous system, it is temptingto speculate that a cross-talk between the immune system andthe nervous system is required not only for normal function butalso development of the immune system. Similarly, less severephenotypes were observed in our mutant compared with thoseof a transgenic mouse model producing anti-NGF neutralizingantibodies (Ruberti et al., 2000) which develops massive celldeath in the spleen, skeletal dystrophy and body weightreduction resulting in mouse lethality. Although that model isof value for other studies (Capsoni et al., 2000), our datasuggest that the non-neuronal phenotypes are most likelycaused by abnormal innervation or a toxic effect of the anti-NGF antibodies on different tissues. Indeed, the massive celldeath observed in the spleen is not easily reconcilable with thelimited expression of NGF receptors in this organ.

NGF has been particularly studied for its effects on Blymphocytes. Several reports have suggested that in vitro NGFcan modulate Ig production as well as B-cell proliferation andsurvival. However, these data often lack consistency and

Development 131 (20) Research article

TrkA +/+ TrkA neo/neo

022B

IgM

1-caM

IgD

022B

CD5

Fig. 6.Aged Tα1-Cre;TrkAneo/neomice have increased levels ofperitoneal B1 cells. Twelve- to eighteen-month-old mutant (n=6) andcontrol (n=6) littermates were sacrificed and peritoneal cells werecollected as previously described (32). Similar numbers of cells wererecovered from both groups. Cells were stained for FACS analysiswith anti-B220, -IgM, -CD5, IgD and -Mac-1 antibodies in triplestaining combinations. Three staining combinations are shown(B220/IgM; Mac-1/IgD; B220/CD5). The B1 cell population isindicated by a box. Percentages of peritoneal B2 cells(B220+/IgMlow/IgDhigh/Mac-1–) and macrophages (B220–/Mac-1+)were not different in mutant mice compared with controls.

5193TrkA function in the immune system

depending on the model used, somewhat contrasting resultswere described (Brodie et al., 1996; Kimata et al., 1991a;Kimata et al., 1991b; Kimata et al., 1991c). Thus, to date it isstill unclear what role NGF exerts in vivo. To a certain degree,the lack of consistency of those results may reflect differencesbetween cell lines. In this study, we have found that althoughB lymphocytes appear to develop normally in the absenceof TrkA, Ig production is affected. Some Ig classes aresignificantly increased. These increases were subtle, but couldbe confirmed in a ‘classical reconstitution model’. Micereconstituted with TrkA-deficient cells showed the same B- andT-cell numbers compared with animals reconstituted withlittermate wild-type cells (not shown). As observed in the‘reverse conditional TrkA mouse model’, we found increasedIg levels in mice reconstituted with TrkA-deficient fetal cells.Importantly, these data validate our genetic model.

It has been reported that antibody neutralization of NGFin vitro and in vivo affects the viability of B memorylymphocytes, suggesting an autocrine role for NGF on thesurvival of memory B lymphocytes (Torcia et al., 1996).Although the mechanism for this effect is still unknown, it hasrecently been suggested that inactivation of p38 MAPK,possibly through a TrkA-activated phosphatase, may berequired to prevent B memory lymphocyte apoptosis (Torcia etal., 2001). In contrast, we have found that TrkA-deficient micecan mount a robust B memory response (Fig. 5A). We testedthe response one and two weeks after the boost dose of antigen,whereas Torcia et al. (Torcia et al., 1996) analyzed the responseafter only four days from the recall-dose of immunogen (sixdays after anti-NGF treatment). Thus, we cannot exclude thatthe discrepancy is caused by different timing of the analysis.Alternatively, we have exclusively ablated TrkA function whileanti-NGF antibodies were used in the previous study.Therefore, it is also possible that other players such as p75 (orother yet unknown NGF receptors) may be involved in thedevelopment of a B memory response. However, ourpreliminary data suggest that p75-deficient mice can alsomount a humoral response.

Following immunization with a T-dependent antigen (KLH-TNP), TrkA-deficient mice are able to produce specific Ig atlevels comparable to those of control littermates. Notably,following immunization the mutants produce more IgG2b, aclass that is overall similarly represented in basal conditions.These data provide definitive evidence that NGF/TrkA does notregulate Ig class switch as previously suggested (Torcia et al.,1996).

In addition to disregulation of Ig levels, we have also foundthat TrkA deficiency causes an increase in peritoneal B1 cellswith aging. Several studies have shown that B1 cells areincreased in mouse models deficient for molecules thatnegatively regulate BCR signaling, whereas B1 are decreasedwhen molecules positively regulating BCR signaling areinactivated (Hardy et al., 2000; Hayakawa and Hardy, 2000).Therefore, it appears that TrkA may play an inhibitory role onBCR signaling. A direct link between BCR- and TrkA-signaling has not yet been described. However, both receptorscan independently affect vav, a Rac guanine nucleotideexchange factor expressed exclusively in hematopoietic cells(Melamed et al., 1999). Thus, it is tempting to speculate thatTrkA may influence BCR signaling by acting on one of itsdownstream players such as vav. This effect on BCR signaling

could ultimately be involved in dictating B-cell commitment toproliferation or differentiation.

Collectively, our data suggest that TrkA, although modulatingimmune system functions, is not required for its normaldevelopment. B- and T-cell number and distribution in spleen,thymus, bone marrow and blood appear unaffected by TrkAdeletion. Similar results have been obtained for mast cells inskin, spleen and lymph nodes despite reports that NGF injectionscause their increase in various tissues of neonatal rats (Aloe andLevi-Montalcini, 1977; Bienenstock et al., 1987; Bullock andJohnson, Jr, 1996). This is a striking result because many studiessuggested that disruption of the NGF/TrkA system in theimmune system would cause dramatic deficits. In our initial invivo studies of TrkA-deficient mast cells, we have been unableto detect a clear phenotype probably because of compensatorymechanisms or vagaries associated with the mixed geneticbackground typical of targeted mutation mouse models (Fig. 3A-F). However, we have detected some abnormalities in theirdegranulation potential in vitro, suggesting that TrkA may havea role in mast cell function (Fig. 3G). Mast cell degranulationfollowed by mediator release is a well-described contributor toairway hyper-reactivity. It has been reported that NGF augmentsthe allergic early-phase reaction in the lung (Path et al., 2002).In vivo experiments using this TrkA mutant mouse model in apure genetic background will help elucidate the role andcontributions of NGF/TrkA in mast cell function and their netcontribution to mast cell-driven pathophysiology. It has beenreported that plasma and/or serum levels of NGF are increasedin several allergic disorders including asthma, urticaria-angioedema, allergic rhinoconjunctivitis and vernalkeratoconjunctivitis (Aloe and Tuveri, 1997; Bonini et al., 1996;Lambiase et al., 1995). Because mast cells express exclusivelyTrkA and not p75, it is tempting to suggest that this receptor mayprovide a target for the management of inflammatory diseases(Micera et al., 2003). Yet, efforts toward this goal have not beenvigorously pursued because the available data had suggested thattampering with the NGF/TrkA pathway would cause severeimmunological deficits. However, the relatively limited impacton immune system development reported here indicatesthat TrkA could provide a useful target for the control ofinflammatory diseases. Thus, this mouse model should providea useful system to test the contribution of NGF/TrkA in thepathology of these disorders.

Finally, this novel approach by ‘reverse conditional genetargeting’, could be applied to many other systems in whichcomplete inactivation of a gene in specific organs/tissues isrequired but cannot be achieved with the currently availablerecombinase and transgenic systems.

We thank Keith Rogers and Diana Heines of the Pathology Histo-Technology Laboratory for preparation and evaluation of thehistological samples, Lynn Laws-Ricker for help and expertise withthe PCA analysis, Mary Ellen Palko and Susan Reid for technicalassistance in the generation and maintenance of the mutant mousestrains, Louise Finch and Refika Turnier for FACS analysis, FredaMiller and Phil Soriano for generously providing, respectively the Tα-1 promoter and the ROSA26 mouse strain, Asher Zilberstein, RobertLewis and George DeSanctis (Aventis Pharmaceuticals), NealCopeland, Nancy Jenkins and all members of the Mouse CancerGenetics Program for helpful suggestions, Esta Sterneck for criticalreading of the manuscript. This work was supported by the NationalCancer Institute, DHHS.

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